Transfer film for laser microcapture

ABSTRACT

The present invention generally provides an improved transfer film having multiple layers for laser micro-capture of a sample. The transfer film for laser micro-capture includes distinct layers for expansion and adhesion in order to optimize the performance of the transfer film. A transfer film including a spring-back layer is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. application Ser. No.09/788,117 filed Feb. 16, 2001 entitled “Transfer film for lasermicrocapture” which claims priority to U.S. Provisional Application No.60/182,832, filed on Feb. 16, 2000 by the same inventors, the entirecontents of all of these applications are hereby incorporated herein byreference as if fully set forth herein.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of lasermicro-capture, and more particularly, to a transfer film for lasermicro-capture of a sample.

[0003] Diseases such as cancer have long been identified by examiningtissue biopsies to identify unusual cells. The problem has been thatthere has been no satisfactory prior-art method to extract the cells ofinterest from the surrounding tissue. Currently, investigators mustattempt to manually extract, or microdissect, cells of interest eitherby attempting to mechanically isolate them with a manual tool or througha convoluted process of isolating and culturing the cells. Mostinvestigators consider both approaches to be tedious, time-consuming,and inefficient.

[0004] A new technique has been developed which can extract single cellsor a small cluster of cells from a tissue sample in a matter of seconds.The technique is called laser capture microdissection (LCM). In lasercapture microdissection, the operator looks through a microscope at abiological specimen such as a tissue biopsy section mounted on astandard glass histopathology slide, which typically contains a varietyof cell types. A capture film is placed over the tissue biopsy section.Upon identifying a group of cells of interest within the tissue section,the operator generates a pulse from a laser. The laser pulse causeslocalized heating of the thermoplastic film as it passes through it,imparting to it an adhesive property. The cells then stick to thelocalized adhesive area of the thermoplastic film directly above them.Upon removal of the film from the biopsy tissue, the selected cells orsections of tissue are transferred along with the film. The film can beextracted in order to remove biomolecules for subsequent analysis.Because of the small diameter of the laser beam, extremely small cellclusters or single cells may be microdissected from a tissue section.

[0005] By taking only these target cells directly from the tissuesample, scientists can immediately analyze the DNA, RNA, proteins, orother biomolecules in order to characterize the activity of the targetcells using other research tools. Such procedures as polymerase chainreaction amplification of DNA and RNA, and enzyme recovery from thetissue sample have been demonstrated.

[0006] Laser capture microdissection has successfully extracted cells inmany types of tissues. These include kidney glomeruli, in situ breastcarcinoma, atypical ductal hyperplasia of the breast, prostaticinterepithielial neoplasia, and lymphoid follicles. The direct access tocells provided by laser micro-capture will likely lead to a revolutionin the understanding of the molecular basis of cancer and otherdiseases, helping to lay the groundwork for earlier and more precisedisease detection.

[0007] Another likely role for the technique is in recording thepatterns of gene expression in various cell types, an emerging issue inmedical research. For instance, the National Cancer Institute's CancerGenome Anatomy Project (CGAP) is attempting to define the patterns ofgene expression in normal, precancerous, and malignant cells. Inprojects such as CGAP, laser capture microdissection is a valuable toolfor procuring pure cell samples from tissue samples.

[0008] The LCM technique is generally described in the publishedarticle: Laser Capture Microdissection, Science, Volume 274, Number5289, Issue 8, pp 998-1001 published in 1996, the entire contents ofwhich are incorporated herein by reference. The purpose of the LCMtechnique is to provide a simple method for the procurement of selectedhuman cells from a heterogeneous population contained on a typicalhistopathology biopsy slide.

[0009] A typical biological specimen is a tissue biopsy sampleconsisting of a 5 to 10 micron slice of tissue that is placed on a glassmicroscope slide using fixation and staining techniques well known inthe field of pathology. This tissue slice is a cross section of the bodyorgan that is being studied. The tissue consists of a variety ofdifferent types of cells. Often a pathologist desires to remove only asmall portion of the tissue for further analysis. Another typicalbiological specimen is a layer of cells coated from a liquid suspension.

[0010] Laser micro-capture employs a transfer film that is placed on topof the tissue sample. The film may contain dyes or pigments chosen toselectively absorb at the frequency of the laser. When the film isexposed to the focused laser beam the exposed region is heated andexpands, contacting and adhering to the tissue in that region. The filmis then lifted from the tissue and the selected portion of the tissue isremoved with the film.

[0011] Transfer films such as a 100-micron thick ethylene vinyl acetate(EVA) film available from Electroseal Corporation of Pompton Lakes, N.J.(type E540) have been used. The film is chosen to have a low meltingpoint of about 60° C.-90° C.

[0012] While the films employed in laser micro-capture applications haveproved satisfactory for the task, a single-layered transfer film hasbeen generally imbued with all of the necessary performancecharacteristics. For example, the transfer film must be capable ofabsorbing the optimum amount of energy from the laser for the desiredactivation of the film. In dye-impregnated films, the optical absorptionis a function of its thickness, the type of dye and concentration of dyeemployed. This property of the film may be in conflict with a desire toselect film thickness for other reasons. The film must also expand adesired amount and be capable of adhering to the specimen in desiredlocations, yet substantially avoid adhesion to undesired particles.Furthermore, it is important to keep the temperature of that portion ofthe transfer film contacting the specimen sufficiently low to avoiddamage to or change in the nature of the specimen. Also, the transferfilm must be capable of being adhered to a carrier and preferably betransparent to enable observation during all stages of the collectionprocedures. These performance characteristics, among others, aredemanded of the transfer film. The present invention is directed toproviding an improved transfer film that de-couples some of theperformance characteristics within the transfer film in order tooptimize the performance of each.

SUMMARY OF THE INVENTION

[0013] In accordance with one aspect of the present invention, there isprovided a transfer film for laser micro-capture of a sample includingat least one expansion layer and an adhesive layer coupled to at leastone expansion layer. The adhesive layer is located between the expansionlayer and a sample for micro-capture. The expansion layer absorbs energyincident upon the transfer film and expands to exert a force upon theadhesive layer such that a selected portion of the sample adheres to theadhesive layer for micro-capture.

[0014] In accordance with another aspect of the present invention, thereis provided a transfer film for laser micro-capture of a sampleincluding at least one expansion layer and an adhesive layer coupled tothe expansion layer. The adhesive layer is located between the expansionlayer and a sample for micro-capture. The expansion layer absorbs energyincident upon the transfer film and expands to exert a force upon theadhesive layer such that the adhesive layer is deflected towards thesample and adheres to a selected portion of the sample. After adhesion,the adhesive layer retracts away from the sample.

[0015] In accordance with yet another aspect of the present invention,there is provided a transfer film for laser micro-capture of a sampleincluding at least one expansion layer, at least one retraction layerand an adhesive layer. The retraction layer is coupled to the expansionlayer, and the adhesive layer is coupled to the retraction layer. Theadhesive layer is located between the retraction layer and a sample formicro-capture. The retraction layer is located between the expansionlayer and the adhesive layer. The expansion layer absorbs energyincident upon the transfer film and expands to exert a force upon theretraction layer and adhesive layer such that the retraction layer andthe adhesive layer are deflected towards the sample and a selectedportion of the sample adheres to the adhesive layer for micro-capture.After adhesion, the retraction layer with the attached adhesive layerretracts away from the sample.

[0016] In accordance with yet another aspect of the present invention,there is provided a transfer film for laser micro-capture having a firstlayer and a second layer. The first layer is thermally coupled to atleast a first energy-absorbing substance selected to absorb energywithin a first spectrum. The second layer is thermally coupled to atleast a second energy-absorbing substance selected to absorb energywithin a second spectrum. The first layer provides a first expansionupon activation by at least a first laser pulse of energy within thefirst spectrum to exert a force on the second layer such that a portionof the second layer is moved towards the sample at least a firstdistance. The second layer provides a second expansion upon activationby at least a second laser pulse of energy within the second spectrumsuch that the portion of the second layer moves towards the sample asecond distance.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The foregoing and other advantages of the invention will becomeapparent upon reading the following detailed description and uponreference to the drawings in which:

[0018]FIG. 1 is a side-elevational view of an apparatus for lasermicro-capture;

[0019]FIG. 2 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0020]FIG. 3 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0021]FIG. 4 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0022]FIG. 5 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0023]FIG. 6 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0024]FIG. 7 is a side view of a transfer film, carrier, and sample ofthe present invention;

[0025]FIG. 8 is a side view of a transfer film, carrier, and sample ofthe present invention; and

[0026]FIG. 9 is a side view of a transfer film, carrier, and sample ofthe present invention.

[0027] While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and described in detail herein. However, itshould be understood that the invention is not intended to be limited tothe particular forms disclosed. Rather the invention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The present invention and the various features and advantageousdetails thereof are explained more fully with reference to thenonlimiting embodiments that are illustrated in the accompanyingdrawings and detailed in the following description. Descriptions ofwell-known components and processing techniques are omitted so as not tounnecessarily obscure the present invention.

[0029] Turning now to the drawings and referring initially to FIG. 1,there is depicted a transfer film 10 coupled to a substrate or carrier12 in the shape of a cap. The carrier or cap 12 is adapted for abiological analysis vessel of the type disclosed in U.S. Pat. No.5,859,699 issued on Jan. 12, 1999 entitled “Laser CaptureMicrodissection Analysis Vessel”, U.S. Pat. No. 6,157,446 issued on Dec.5, 2000, entitled “Laser Capture Microdissection Analysis Vessel”, U.S.Pat. No. 5,985,085 issued on Nov. 16, 1999 entitled “Method ofManufacturing Consumable for Laser Capture Microdissection”, U.S. Ser.No. 081984,979 filed on Dec. 4, 1997, and U.S. Ser. No. 09/357,423 filedon Jul. 20, 1999 all of which are incorporated herein by reference intheir entirety.

[0030] The cap 12 is made from an inert and, preferably, transparentplastic such as acrylic (polymethyl methacrylate). The carrier 12 isshaped as a cap and adapted to be removably coupled to an analysisvessel such as a centrifuge tube, microtiter plate, or other well-knownvessels. The cap 12 has an upper portion 14 and a lower portion 16. Theupper portion 14 includes a top surface 18 and a shoulder 20. The cap 12may be provided with an identifying serial number such as a bar codelabel or laser-etched label that provides for easy identification andtracking of cell samples. The lower portion 16 includes a substratesurface 22 to which the transfer film 10 is coupled. The cap 12 and itsconfiguration are not limited to this geometry.

[0031] The cap 12 of FIG. 1 is easily handled either manually or byautomated means such as an LCM apparatus of the kind disclosed in thefollowing co-pending applications: U.S. Ser. No. 09/018,452 filed Feb.4, 1998, U.S. Ser. No. 09/121,691 filed on Jul. 23, 1998, U.S. Ser. No.09/121,635 filed on Jul. 23, 1998, U.S. Ser. No. 09/058,711 filed onApr. 10, 1998, U.S. Ser. No. 09/121,677 filed on Jul. 23, 1998, U.S.Ser. No. 09/208,604 filed on Dec. 8, 1998, and U.S. Ser. No. 09/617,742filed on Jul. 17, 2000 the entire contents of all of these applicationsare hereby incorporated by reference in their entirety as if fully setforth herein. The cap 12 facilitates obtaining the sample and decreasesthe possibility of DNA contamination of the sample during handling andtransport.

[0032] The cap 12 is shown positioned over a glass slide 24 and a tissuesample 26. The glass slide 24 and cap 12 are placed under a microscopeobjective and a laser pulse, shown diagrammatically at reference numeral28, is directed at a selected region of the tissue sample 26 to performthe laser capture microdissection. Those of ordinary skill in the artwill appreciate that an alternate configuration that may be employed isan inverted microscope wherein the tissue sample 26 may be viewed fromunderneath the sample slide 24. Such skilled persons will appreciatethat the present invention may easily be used in such a configuration.

[0033] Suitable lasers for use in the present invention include carbondioxide lasers (9.6-11 micrometer wavelengths), laser diodes, tunablesingle frequency titanium sapphire lasers, and diode-pumpedneodymium-doped, yttrium-aluminum garnet (Nd:YAG) lasers. The wavelengthoutputs from these lasers can preferably range from ultraviolet toinfrared. A particularly desirable laser for use with the presentinvention is a laser diode with wavelengths between approximately 690 nmand 1300 nm. In this wavelength range, conventional glass microscopeoptics are highly transmissive and can be used to focus the laser.

[0034] The laser capture operation can be simply described. First, themicroscope stage is centered and the transfer film 10 coupled to the cap12 is placed in position above the tissue sample 26 on slide 24. Thetransfer film 10 contacts the tissue sample 26. Next, the tissue sample26 is inspected until desired cells are located. Then, the laseractivates the transfer film 10, which absorbs energy from the laser. Asa result, a selected portion of the transfer film 10 expands to contactthe tissue and cause adhesion of the target cells to the transfer film10. Alternatively, the transfer film 10 is spaced from the sample 26(noncontact laser micro-capture). In this case, the expansion of thetransfer film 10 upon activation by the laser pulse 28 will simplyinject a portion of the transfer film 10 into the tissue sample 26 forcapturing target sample cells. Mechanical adhesion occurs asinterlocking occurs when, due to heating, the thermoplastic materialflows about and into the voids of the rough tissue sample surface andinterlocks upon subsequent cooling.

[0035] In contact micro-capture, the transfer film 10 makes contact witha tissue section prior to activation by the laser pulse 28. Due to thefriable nature of tissue sections, loose material (whole cell ormacromolecular) is likely to adhere to the transfer film 10 even thoughthe laser did not illuminate them resulting in non-specific transfer ofmaterial. If these portions are transferred to the reagent vessel, theywill be digested by the reagents and appear as contaminants in thesample. It is important to prevent the loosely bound tissue areas fromcontacting the film 10. Reducing this problem by providing a non-stickbarrier layer in contact micro-capture is described in copendingapplication U.S. Ser. No. 09/562,495 filed on May 1, 2000, which is, inits entirety, incorporated herein by reference. Another way of reducingnon-specific transfer, for example, is non-contact LCM. In non-contactLCM, the transfer film is offset or distanced a few microns from thetissue sample as described in co-pending application U.S. Ser. No.08/984,979 filed on Dec. 4, 1997.

[0036] After the activated portion of the transfer film 10 solidifies,the film 10 is withdrawn. The physical interface between the transferfilm 10 and the selected cell area of the sample intended formicrodissection causes the transfer film 10 when it is withdrawn to“pull” the selected sample area from the remainder of the specimen.Micro-capture occurs.

[0037] For laser micro-capture, the transfer film 10 is adapted forabsorbing energy delivered by the laser pulse 28 or multiple pulses ofthe same or different energy wavelengths. The transfer film 10 isfurther adapted for expanding and adhering to the target cells.Typically, a single layer within the transfer film 10 performs all ofthe functions of energy absorption, expansion and adhesion; and asuitable material having all of the desired characteristics is selected.According to one aspect of the present invention, the transfer film 10includes more than one layer such that one or more of the functions ofabsorption, expansion and adhesion are de-coupled into one or moreseparate layers. By separating one or more of the functions intoseparate layers within the transfer film 10, the performance of transferfilm 10 is increased by optimizing the materials selected to performeach function or combination of functions.

[0038] A variety of thermoplastic polymer films is widely used as heatactivated adhesives that are suitable for the transfer film 10. It ispreferable to use a polymer film having a high melt index range such asgreater than 100 dg/min so that it is activatable at lower temperaturesto avoid damage to or change in the nature of the tissue sample 26.Therefore, it is important that the temperature of the portion of thetransfer film contacting the sample is below approximately 100° C.,preferably below approximately 80° C., and more preferably belowapproximately 60° C. Melt index is measured according to ASTM D1238 inwhich a sample of polymeric material is melted isothermally in a heatedchamber and then pushed out of a capillary orifice under a fixed load.The amount of extruded material over time is measured and the melt flowrate (index) is determined in decigrams/minute.

[0039] Turning now to FIG. 2, a cross sectional view of a transfer film100 is shown. The transfer film 100 includes an expansion layer 112having a first surface 111 and a second surface 113. The transfer film100 is coupled to a substrate cap 116 at the first surface 111 and to anadhesive layer 114 at the second surface 113 such that the expansionlayer 112 is located between the adhesive layer 114 and the substratecap 116. The adhesive layer 114 includes a first surface 115 and asecond surface 117. The thickness of the transfer film 100 isapproximately greater than approximately 10 μm.

[0040] The expansion layer 112 includes a material adapted for expansionthat is made from a wide variety of electromagnetically or thermallyactivatable materials. For conventional one layer micro-capture, thepolymer must be chosen such that it has “hot-melt” characteristics, inthat it becomes adhesive as its temperature is raised. In particular,ELVAX™ 410, 200 and 205 are suitable resins of EVA that are commerciallyavailable from DuPont E. I. de Nemours and Company of Wilmington, Del.Generally, the EVA should have a high-melt index indicated by a lowviscosity and low molecular weight. EVA, among plastics, has a uniquelylow melting range, which can be controlled by its manufacture. Suchmanufacture control can include the addition of a variety of ingredients(e.g. co-polymers) to adjust the melting point and other properties ofthe EVA. Persons of ordinary skill in the art will recognize that othermaterials having desirable properties may also be employed.

[0041] The said limitation of needing a polymer that simultaneously actsas an energy-adsorbing medium, expands, wets and adheres to the sample,does not hold for the expansion layer of the present invention, thusallowing any polymer that merely expands with the absorption of energyto be used. This allows the use of essentially any polymeric materialwhich can absorb the energy or contains an energy adsorbing substance,and which can melt-flow during the application of a laser pulse. Theseinclude polymers such as one or more of the following: silicone,polyimides, polyesters, polyethers, fluoropolymers, polyethyelene andit's copolymers such as ethylene vinyl acetate (EVA), polyurethanes,polyvinyl acetates, ethylene-methyl methacrylate, polycarbonates,ethylene-vinyl alcohol copolymers, polypropylene, and expandable orgeneral purpose polystyrenes. Additives to modify the base polymersincluding plasticizers, antioxidant stabilizers, pigments or dyes can beused as known to the persons skilled in the art.

[0042] The expansion layer 112 comprising EVA, for example, expandsisotropically when it is exposed to the energy from the laser. As anapproximation, the EVA film expands approximately 12-15% downward andupward when it is exposed to the charge from the laser. The substratecap 116 may restrict upward expansion. The material used as theexpansion layer 112 can be an EVA of the type used for conventionallaser micro-capture, but the restriction on melt index and melttemperature is not as great in the present invention because theexpansion layer does not have to come into direct contact with thebiological sample. Hence, expansion and adhesion are de-coupled in thetransfer film of the present invention such that expansion is providedby the expansion layer 112 and adhesion is provided by the adhesivelayer 114. This means that, for instance, EVAs with melt index valuesbelow 200 dg/min could be suitable for the expansion layer and a lowersample contact temperature can be achieved because the expansion layer112 does not contact the sample for adhesion.

[0043] The performance properties for the adhesive layer in thismultilayered construction is wetting and adhesion to the sample. Assuch, several different class of materials can be utilized. In oneembodiment, the adhesive layer 114 in FIG. 2 can be an ordinary pressuresensitive adhesive (PSA), a hot melt adhesive, or a UV- orelectron-beam-curable adhesive or coating. For the PSA category, whichcan be of solvent-based, water-based, or hot-melts subcategories, thepolymeric material can be a rubber (natural, butyl, or styrene-butadienerubber), a block copolymer such as styrene-ethylene/butadiene-styrene(SEBS), styrene-isoprene-styrene (SIS), or other polymers (polybutene,polyvinylether, acrylics, ethylene-vinyl acetate, atactic polypropylene,silicone). The pressure sensitive adhesive may also contain tackifyingagents to increase the adhesiveness of the capture polymer in order tomore effectively microdissect the selected area of tissue withoutcompromising the other properties needed for microdissection. Generally,the tackifying agents must be compatible with the thermoplastic materialto which they are added. The tackifying agents must be at leastpartially soluble and not completely phase separated. As such,tackifying resins such as aliphatic, aromatic, mixed hydrocarbons,rosins, and terpenes can be used. Also, the addition of tackifyingagents must maintain the optical clarity and transparency of the capturepolymer and not compromise the tensile strength to the point thatcohesive failure occurs in the polymer when removing the selectedportion of the tissue. Furthermore, the tackifying agent must also notinterfere with subsequent steps of extraction and molecular analysis ofthe tissue. The amount of tackifying agent is approximately 2 wt. % to50 wt. %, preferably, 8 wt. % to 20 wt. % of the total formulation. Thesoftening point of suitable tackifying agents is between approximately18° C. and 99° C. The softening point of thermoplastics is defined asthe temperature at which the polymers begin to show viscoelasticmovement under particular combinations of load and rate of temperaturerise. Two ASTM methods: D1525 “Test Methods for Vicat SofteningTemperature of Plastics” and E28 “Test Method for Softening Point byRing-and-Ball Apparatus” are commonly used. In addition to the importanttackifying agents described, common additives, such as plasticizers,antioxidant stabilizers, thioxotropic agents, coupling agents, UVabsorbers, and pigments or dyes can be employed. For the hot meltadhesives category, polymeric material can be EVA copolymers, acidterpolymers, EMA copolymers, ethylene n-butyl acrylate copolymers, lowdensity polyethylene, polypropylene, polyisobutylene, polybutene,polyamides, or sulfonated branched polyester. Tackifying agents andadditives are the same as described above. UV-curable adhesives can beeither free-radically or cationically initiated, and most commonly arebased on acrylates, methacrylates, or epoxy-based. EB-curable adhesivesare most commonly based on vinyl chemistry, optionally in combinationwith pro-radiation additives.

[0044] As an example of a case where the adhesive layer 114 is apressure sensitive adhesive, this layer is preferably un-doped such thata low sample contact temperature upon adhesion can be maintained. Thethickness of the adhesive layer 114 is approximately 0.1-10 μm,preferably less than approximately 5 μm.

[0045] Generally, the pressure sensitive adhesive material is coupled toan expanding, heat-activated portion of the transfer film 100 such asthe expansion layer 112. The expanding portion of the expansion layer112 pushes on the pressure sensitive adhesive material such thatsufficient pressure is exerted in contact with the tissue sample 118.Application of sufficient pressure causes the pressure sensitiveadhesive to flow. When the pressure is removed, the melt strength of thepolymer is high enough to hold and adhere to the target cells which aresubsequently excised or captured. One advantage of using a pressuresensitive adhesive within the adhesive layer 114 is that the adhesivelayer 114 that comes into contact with the tissue sample 118 is not ashot as the expanding layer 112. This advantage provides for a moregentle capture and may facilitate the capture of live cells.

[0046] The expansion of the polymer occurs through several mechanisms.One mechanism is the melting of crystallites of a semicrystallinepolymer, another mechanism is the solid to liquid transition, a thirdmechanism is the simple bulk expansion of a material with temperaturerepresented by the thermal coefficient of expansion, and a fourthmechanism is the generation of gas within the expansion layer.

[0047] One benefit of the generation of gas bubbles is the increasedexpansion that they provide over the expansion due to the polymersolid-solid, solid-liquid, and bulk thermal expansions. The gas can befrom expansion of the small amounts of small molecules in the film(water, residual solvents, dissolved gasses, etc.), volatilization ofthe polymer itself, and/or from molecules generated from thermaldegradation. Blowing agents can be added to the film in order tofacilitate gas generation. The term blowing agent is used to refer to apolymer additive, which generates gas bubbles in the polymer. Theseagents are commonly used to create foamed polymers. “Physical” foamingagents are compounds, which expand within the polymer without anassociated chemical reaction. An example of a physical foaming agent isa small molecule, commonly a hydrocarbon, fluorocarbon, or chlorocarbonwith a relatively low boiling point, which is dissolved into a polymer,and is then induced to foam by the introduction of thermal energy and/ora reduction of pressure. “Chemical” foaming agents generate gasses via achemical reaction, usually resulting in the generation of gasses such asnitrogen, CO₂, hydrogen, etc. Examples of chemical foaming agents areazo compounds, hydrazides, peroxides, and carbonates. Some of thesematerials decompose exothermically, some decompose endothermically.Specific compounds which can be used are azodicarbonamide andderivatives, sulfohydrazides such as 4,4′oxybisbenzenesulfonylhydrazide, sodium salts of carbonic acid, and 5-phenyl tetrazole. Thechemical foaming agents generate gasses when exposed to energy, usuallyin the form of heat or light.

[0048] Blowing agents are advantageous in that the gas is generated in acontrolled manner at a controlled temperature. It is desired to generatethe gas in a small region in order to perform laser capture in a smallspot. By generating the gas at a relatively low temperature, the bubblecan be formed at relatively low laser power, and with a relatively shortpulse. The short pulse allows the heat to create the bubble in a smallregion before thermal diffusion can expand that region undesirably farfrom the irradiation region.

[0049] One embodiment of the present invention is to have at least onelayer of the transfer film 100, such as the expansion layer 112, containa thermally activated foaming agent thermally coupled to anenergy-absorbing substance of the type discussed below, including aninfrared absorbing dye of the type discussed below. The dye, forexample, absorbs energy from the laser causing heating of the expansionlayer polymer, which also causes the blowing agent to generate gas inthe film 100. Alternatively, the dye can be in a layer (not shown)separate from the blowing agent. In some cases, it is advantageous tolocate the layer, such as the expansion layer 112, containing theblowing agent between the adhesive layer 114 and the cap 116 such thatthe bubble formed by the generated gas pushes the adhesive layer 114into the tissue below. If a photochemically activated blowing agent isused, the need for a separate dye for heat absorption can be reduced oreliminated.

[0050] Still referencing FIG. 2, the transfer film 100 is also adaptedfor energy absorption such that at least one layer of the transfer film100 absorbs energy incident upon the transfer film 100 such as energyfrom the laser beam or other activating light source to activate thetransfer film 100. A variety of wavelengths of electromagnetic energycan be used in the practice of the invention provided that suitablematerials are used. In particular, it is important that the transferlayer 100 absorbs sufficient energy at the chosen wavelength orwavelengths to provide expansion of at least the expansion layer 112 inthe targeted region as well as to impart any desirable adhesioncharacteristics to the adhesion layer 114. For a transfer film 100comprising thermoplastic materials such as EVA, a wavelength ofapproximately 0.3 μm to approximately 10.0 μm is preferred as thesematerials intrinsically absorb in this range. It is preferred that thewavelengths for laser activation and energy absorption be chosen outsidethe normal range used for microscopic imaging. For example, a variety ofwavelengths from the laser can be employed for reproduciblemicrotransfer of tissue.

[0051] To enhance energy absorption, the transfer film 100 can includean energy-absorptive substance. For example, the expansion layer 112 maybe thermally coupled to an energy-absorptive substance and/or theadhesive layer 114 may be thermally coupled to an energy-absorptivesubstance. Thermal coupling merely requires that heat in one of thelayer or layers is capable of being directly or indirectly transportedto another layer or layers. As is well known, thermal transport can beachieved by conduction, convection, or radiation.

[0052] There are many well-known energy-absorptive substances that arecapable of being thermally coupled to the transfer film 100 either tothe expansion layer 112 or adhesive layer 114 or both. For example, theenergy-absorptive substance can include an absorptive dye. This dye canbe either a broadband absorptive dye or a frequency-specific absorptivedye. A broadband absorptive dye is one capable of absorbing a portion ofthe electromagnetic spectrum, preferably a portion within a range havinga wavelength of approximately 0.3 μm to approximately 10.0 μm. Thebroadband absorptive dye can have a relatively broad absorption line orabsorb energy throughout the visible region of the spectrum so that thedye does not affect the color spectrum of the transmitted light that isused to illuminate the sample. Broadband absorptive dye can providestrong absorption of a range of wavelengths without altering thetransparency of the transfer film 100 to visible light. Generally, theexpansion layer 112 has a high dye level (optical density>0.4).

[0053] It is also possible to thermally couple infrared absorbing dyesto the transfer film 100 to provide strong absorption at other specificinfrared wavelengths without altering the films transparency to visiblelight. Such dyes are preferably infrared absorbing dyes, which arereadily soluble in plastic films and have a high extinction coefficient,narrow infrared or near-infrared absorption (e.g. from 750 to 1500 nm,and more particularly from 750 to 1100 nm) bands that can be matched toa variety of infrared or near-infrared lasers (including laser diodes).If the focused pulse of electromagnetic radiation (e.g., a laser pulse)is delivered at wavelengths that are strongly absorbed by the film, thenthe film may be efficiently activated.

[0054] The response of the system described here may involve temporaldependence as well. The thermo-mechanical features may be optimized inlight of pulsing at least a first laser to achieve the appropriatechange in volume of the first expansion layer 112 whereupon a secondlaser is pulsed to activate a spectrally separate layer such as athermally coupled adhesive layer 114. This multiple pulse technique isutilized to provide broad area, temporary expansion of the expansionlayer 112 coupled with a spatially selective (approximately 1 μm to 10μm expansion of the adhesive layer 114. The expansion layer 112 wouldthen relax, effecting capture of the desired tissue. Film thickness andabsorbance are tailored for optimal temporal, optical and spectralperformance. The correspondingly appropriate pulses of the correctwavelength, duration and spot-size would address these properties.

[0055] For example a first layer such as the expansion layer 112 isthermally coupled to at least a first energy-absorbing substanceselected to absorb energy within a first spectrum. The second layer suchas the adhesive layer 114 is thermally coupled to at least a secondenergy-absorbing substance selected to absorb energy within a secondspectrum. The first layer provides a first expansion upon activation byat least a first laser pulse of energy within the first spectrum toexert a force on the second layer such that a portion of the secondlayer is moved towards the sample at least a first distance. The secondlayer provides a second expansion upon activation by at least a secondlaser pulse of energy within the second spectrum such that the portionof the second layer moves towards the sample a second distance. Hence,any one or more layers such as the expansion layer 112 and/or theadhesive layer 114 may be thermally coupled or doped with at least oneindependently addressable, spectrally selective, energy absorbingsubstance.

[0056] Many types of dyes could be considered for infrared absorption,since most classes of dyes that absorb energy having wavelengths in thevisible spectrum can be extended in range of wavelength absorption bymolecular modification. Phthalocyanines, cyanines and Epolight 4019 fromEpolin Inc., of Newark, N.J., have been among the most popular dyesbecause of stability, ease of preparation, solubility, optical and otherproperties including narrow band, high extinction coefficient and hightemperature plastic processing. Moreover, the number of possiblemodifications of these dyes is very large because various central metalatoms which can be added and a variety of ring attachments, which can bemade to them. Naphthalocyanine dyes are examples of near-infraredabsorbing dyes.

[0057] Some examples of naphthalocyanine absorptive dyes include one ormore of the following: tin(IV) 2,3-naphthalocyanine dichloride;silicon(IV) 2,3-naphthalocyanine dihydroxide; silicon (IV)2,3-naphthalocyanine dioctyloxide; and vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine. Vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, for example, absorbsnear infrared with a narrow absorption peak at 808 nm which closelymatches the emission wavelength of laser diodes made of gallium arsenidewith aluminum doping (AlGaAs) widely used to pump solid state Nd:YAGlasers. Some of the naphthalocyanine dyes are moderately soluble in EVApolymers and other similar thermoplastic materials. They are stablecompounds with heating to approximately 300° C. and do not exhibitadverse photochemistry, which might affect biological macromolecules inthe tissue.

[0058] In the case where the energy-absorptive substance is anenergy-absorptive dye, the transfer film 100 is doped with the dye usingtechniques well known in the art. For example, the dye may be mixed withthe melted bulk plastic at an elevated temperature and then manufacturedinto a film using standard film manufacturing techniques. The doped filmitself may then constitute the transfer film 100 or the doped film ofthermoplastic may be thermally coupled as a separate layer such as theexpansion layer 112 or adhesive layer 114 or both to form the transferfilm 100.

[0059] Alternatively, organic solvents incorporating dye may be used todope the expansion layer 112 and/or adhesive layer 114 by immersion intothe solvent followed by diffusion of the dye-containing solvent. Theconcentration of dye in solution and the duration of immersion can beadjusted to obtain the desired concentration and depth of diffusion ofthe dye. Generally, the concentration of dye will result in a gradientof dye concentration such that the concentration of dye will decrease ina direction inward from the surface where the dye concentration will begreater relative to the concentration of dye inwards from the surface.The solvent can then be removed, for example, by vacuum degassing.

[0060] Alternatively, a coating material may be applied to the transferfilm. For example, dye-containing solvent may be applied to theexpansion layer 112 and/or adhesive layer 114 and followed byevaporation of the solvent. The concentration of dye may also form agradient in a direction away from the surface of the coatedthermoplastic as it penetrates into the film with a depth determined bythe duration and amount of solvent applied to the film. Coatings on thelow-temperature film surfaces can be applied by spraying, dipping, orspreading. To apply the coatings evenly it may be necessary to preparethe surface of the thermoplastic by plasma etching to make it amenableto solution wetting and provide satisfactory film adhesion.

[0061] Various naphthalocyanine compounds, for example, are soluble insolvents such as methylene chloride or chloroform. Dissolving a dye intoa solvent to form a dye coating is also possible with a metalnaphthalocyanine compound that is obtained by synthesizing a metal-freenaphthalocyanine compound and introducing a metal therein. This processenables introduction of almost all metals and a choice may be made froma wide variety of metal naphthalocyanine compounds. The naphthalocyaninecompounds have a maximum absorption wavelength in the range of 550 to1100 nm. Thus, they find use as photo-functional materials accommodatingthe wavelength of certain lasers.

[0062] The transfer film 100 may include other absorptive substancesincluding non-dye materials that are thermally coupled to the expansionlayer 112 and/or adhesive layer 114. Non-dye energy absorbers include aplurality of Fullerines (i.e.; Bucky Balls, e.g., C60), or a metal filmof nichrome or titanium. The transfer film 100 may include the metalfilm via doping or as a separate layer that is thermally coupled to theexpansion layer 112 and/or the adhesive layer 114. For example, metalfilms of nichrome or titanium can be deposited on a surface which isthen attached to the thermoplastic by first evaporating a very thinlayer of metal film (approximately 10-100 Å) onto a transparent supportfilm such as mylar or polyester using a deposition technique such assputtering. The deposition is halted when the appropriate absorptionlevel of the film is reached. This is a procedure that is well known tothose skilled in the art of thin film coating. If necessary, thethermoplastic material, such as EVA, may be dissolved in a solvent suchas methylene chloride to reduce its viscosity as is well known in theart. Buckminsterfullerene, available as product #379646 from SigmaChemical Company of St. Louis, Mo., can also be used to dope thethermoplastic by mixing the Buckminsterfullerene with heated EVA, forexample, in a concentration that provides the desired energy-absorption.Broadband absorbers are discussed in co-pending application U.S. Ser.No. 08/800,882 filed on Feb. 14, 1997 which is incorporated herein byreference in its entirety. Furthermore, a polymer nanocomposite,containing thermally conductive nanoparticles (e.g., nano alumina,nano-boron nitride, etc.) can be employed in which the filler particlesize is at most 25% of the visible wavelength, such that lightscattering is minimized, leading to a highly transparent, yet thermallyconductive polymeric film.

[0063] As described above, the adhesive layer 114 is activatable andadheres to the sample 118 when activated by the laser beam. The layerserving as an adhesive layer 114 may contain one or moreenergy-absorptive substances of the type discussed above and thermallycoupled thereto in the manner discussed above with respect to theexpansion layer 112. Alternative to such homogenous distribution of dyeparticles in the expansion and/or adhesive layers described in FIG. 2,the adhesive layer 114 may be doped with energy-absorbing dye such thata concentration gradient 122 is formed within the layer 114 as shown inFIG. 3. Here, the adhesive layer 114 is shown to be doped, for example,with a dye containing solvent such that the dye diffuses into theadhesive layer 114 at the second surface 117. The invention is not solimited and the adhesive layer 114 can be doped at the first surface 115and/or second surface 117. Similar to such concentration gradientsrelated to the adhesive layer, a concentration gradient can be employedin the expansion layer 112. This can be achieved by doping the expansionlayer 112 with a dye-containing solvent such that the dye diffuses intothe expansion layer 112 at the first surface 111. The invention is notso limited and the expansion layer 112 can be doped at the secondsurface 113. Moreover, the expansion layer 112 is thermally coupled toan energy-absorbing substance at its first surface 111 and/or its secondsurface 113. Different methods of thermally coupling the expansion layer112 to an energy-absorbing substance include doping to form aconcentration gradient, doping to form a substantially uniformconcentration, coupling a separate energy-absorptive layer that may bedoped or un-doped as discussed above are within the scope of the presentinvention. Any combination of doping the first and/or the secondsurfaces 111, 113 is thus within the scope of the invention.Furthermore, the expansion layer 112 has a softening point ofapproximately 60° C. to approximately 150° C. and a thickness ofapproximately 10 μm to 80 μm The softening point of the adhesive layer114, which comes into contact with the sample, is generally less thanthe softening point of the expansion layer 112, and preferably less than90° C.

[0064] With regards to the method of making or manufacturing themultilayered transfer films of the current invention can be divided intoseveral categories. In the first, conventional polymer processingtechniques such as co-extrusion and lamination, the latter can beoperated continuously as in a calendaring process, or as a batch-processas in compression molding. For example, the transfer film 100 can bemanufactured by passing two distinct layers 112 and 114 throughlamination rollers. In addition to these melt processes, a thin layer(for example, that of an adhesive layer) can be solvent coated ontoanother layer (for example, the expansion layer). These solvent-basedprocesses include, but are not limited to, spray coating, gravurecoating, and dip coating.

[0065] In addition to a multilayer construction, the current inventionis not so limited, and it is understood that a transfer film 100 havingan expansion layer 112 and an adhesive layer 114 can be formed viadoping a single layer in such a manner as to effect dual layerperformance using energy-absorbing substances and methods describedabove. This is illustrated in FIG. 4. Here, the transfer film 100 is asingle layer of at least one polymer of the type discussed above inreference to the expansion layer 112 that is doped with at least oneenergy-absorbing substance of the type and in the manner described abovesuch that an expansion layer 112 and an adhesive layer 114 are therebyformed. Although FIG. 4 illustrates doping from a first surface 111 suchthat a concentration gradient 120 is formed with a high concentration ofdopant at the first surface 111 relative to a second surface 117, theinvention is not so limited and a more uniform concentration ofenergy-absorbing substance can be formed. Basically, the concentrationof energy-absorbing substance in the expansion layer 112 can be tailoredin shape as well as concentration. Although the expansion layer 112 inFIG. 4 is readily discernable, the expansion layer 112 does not have adistinct second surface 113 separating the expansion layer 112 from theadhesive layer 114 nor does the adhesive layer 114 have a distinct firstsurface 115 as in FIGS. 2 or 3. An energy-absorptive substance need notbe employed. Instead, dual-layer performance can be achieved byfabricating a single layer of one or more polymers of the type describedin reference to the expansion layer 112 above.

[0066] With regards to the choice of a polymeric material for thissingle-layer construction, once again, one is limited to a material thatsimultaneously acts as an energy-adsorbing medium, expands, wets andadheres to the sample, for example an EVA material with relatively highmelt index. Moreover, as mentioned above, EVA or other thermoplasticscan be doped by applying a solvent containing a suitable dye to one sideof the transfer film 100. The dye will penetrate into the thermoplasticfilm with a depth determined by the duration, amount of solvent appliedto the film, and the concentration of dye within the solvent. Theconcentration of the dye and the amount of solvent can be adjusted toprovide a concentration gradient of a specific magnitude. The dye isgenerally dissolved into the solvent at a concentration of approximately0.001 wt. % to 1 wt. %. The solvent is chosen such that it completelydissolves the dye in the desired concentration range, and that it swellsthe polymer. For the polymers described herein, with naphthalocyaninedyes, solvents such as methylene chloride chloroform, toluene, andcyclohexane are suitable. It is most convenient to operate at roomtemperature. With the solvent and polymers described herein, the timesneeded will be from seconds to hours depending on the desired amount ofdye doping. In many cases, dye penetration on the order of approximately10 μm to 20 μm is desired.

[0067] Another method of doping thermoplastic film is to place a barrierthat is impenetrable to the dye-containing solvent on one side of thefilm, leaving the other side accessible to the solvent/dye solution. Thefilm can then be dipped into the solvent/dye solution and left for anappropriate amount of time to develop a specific gradient within thethermoplastic. The concentration of the dye in the solvent, the type ofsolvent and its aggressiveness to EVA, the temperature of the solventand the amount of time the film is in contact with the solution can allbe adjusted to provide the appropriate gradient. The solvent can then beremoved, for example, by vacuum degassing.

[0068] When activated by a laser beam, the expansion layer begins tosoften and expand in a direction toward the sample 118. Any expansion ofthe expansion layer 112 in a direction toward the substrate cap 116 iscontained by the rigid cap 116. Hence, a force by the expandingexpansion layer 112 is exerted upon the adjacent adhesive layer 114. Theamount of force exerted by the expansion layer 112 can be customized byselecting materials and thicknesses for the desired mechanical andthermal response.

[0069] The response of the system described here may involve temporaldependence as well. The thermo-mechanical features may be optimized inlight of pulsing a first laser to achieve the appropriate expansion ofthe expansion layer 112 whereupon a second laser is pulsed to activate aspectrally separate layer such as a thermally coupled adhesive layer114. This technique could be utilized to provide a broad area temporaryexpansion of the expansion layer 112 coupled with a spatially selective(i.e. 1-10 micron level) expansion of the thermally coupled adhesivelayer 114. The expansion layer 112 would then relax, effecting captureof the desired tissue. Film thickness and absorbance are tailored foroptimal temporal, optical and spectral performance. Hence, any one ormore layers such as the expansion layer 112 and/or the adhesive layer114 may be doped with at least one independently addressable, spectrallyselective energy-absorbing substance.

[0070] Referring now to FIGS. 5-7, a cross sectional view of a transferfilm 200 attached to a cap 216 is shown. The transfer film 200 includesan expansion layer 212 and an adhesive layer 214. The expansion layer212 includes a first surface 211 and a second surface 213. The expansionlayer 212 is coupled to a substrate cap 216 at the first surface 211 andto the adhesive layer 214 at the second surface 213 such that theexpansion layer 212 is located between the cap 216 and the adhesivelayer 214. The adhesive layer 214 includes a first surface 215 and asecond surface 217. The thickness of the transfer film 200 is greaterthan approximately 10 μm with the expansion layer 212 having a thicknessof approximately 10-80 μm and the adhesive layer having a thickness ofapproximately 0.1-10 μm preferably less than 5 μm.

[0071] The transfer film 200 is similar to the transfer film 100described in FIGS. 2-4; however, the adhesive layer 214 includesadditional retractive properties. The expansion layer 212 is a layerthat expands when activated by the laser beam. The expansion layer 212is capable of absorbing a sufficient amount of energy to expand andflow; and thereby, push upon a substantially unmelted adhesive layer 214that is preferably not activated by a laser pulse 228. In oneembodiment, a portion of the adhesive layer 214 that is pushed by theexpansion layer 212 contacts the sample and adheres thereto as shown inFIG. 6. Hence, the adhesive layer 214 also provides adhesive qualitiesfor microdissection. Upon adhesion to the sample 218, the adhesive layer214 is partially retracted towards the substrate cap 216 by a retractiveforce 226 exerted by the unmelted adhesive layer 214 as shown in FIG. 7.Hence, the adhesive layer 214 also provides retraction qualities thatenhance the mechanical response of the transfer film 200. After thetarget cells are adhered, it is desired that the transfer film 200retract from the tissue sample 218 pulling the cells out of the sample.The thickness of the expansion layer 212 is approximately 20 82 m to 30μm. The thickness of the adhesive layer 214 is approximately 0.1-10 μm,preferably less than 5 μm It is understood that the expansion layer 212may be thermally coupled to at least one energy-absorbing substance ofthe type and in the manner discussed above. For example, thermalcoupling can be accomplished by doping the layer 212 with one or moreenergy absorbing substances or by attaching an energy-absorbing layer ofthe type and the manner discussed above with respect to the expansionlayer 112.

[0072] From a materials point of view, the choice of expansion layer 212can be identical to its counterpart 112 of FIGS. 2-3 (as describedabove). The adhesive layer 214 is similar to the adhesive layer 114 ofFIGS. 2-3 (i.e., it needs to effectively wet and adhere to the sample),however, additionally it must have retractive properties. As such, oneneeds an adhesive polymeric system that has unique viscoelasticproperties. In particular, one needs an elastomeric material thatcontains partial cross-links, which in turn can be present either aschemical cross-links (e.g., silicone elastomers), or as physicalcross-links (e.g., block copolymers). Chemical cross-linked systemsinclude, but not limited to silicone elastosmers, polyurethanes,polyureas, epoxies, various electron-beamed polymers (polyethylenes,polypropylenes, fluoropolymers), peroxide cross-linked vinyl-containingpolymers. Physical cross-linked systems, include polymers such as, butnot limited to rubbers (natural, butyl, or styrene-butadiene rubber),block copolymers such as styrene-ethylene/butadiene-styrene (SEBS),styrene-isoprene-styrene (SIS), polyisobutylene, and polybutenes can beemployed. As before, various compatible tackifying agents in low tomoderate ratios, as well as other commonly, used additives, known tothose skilled in the art of polymer formulation can be employed toachieve desired balance of materials properties. The adhesive layer 214may be thermally coupled to at least one energy-absorbing substance asdescribed above in reference to the adhesive layer 114. Preferably, theadhesive layer 214 is undoped such that it is not activated by the laserpulse and does not substantially plastically deform so that it maysubstantially elastically retract away from the sample 218. If the laserpulse is short enough to prevent heat deposited into the expansion layer212 to propagate into the undoped adhesive layer 214, the adhesive layer214 will not melt.

[0073] The basic mode of constructing and manufacturing the transferfilm 200 is, as a first approximation, identical to that of the transferfilm 100, as described above. The adhesive layer 214 can be doped in ahomogenous fashion with at least one energy-absorptive substance of thetype and in the manner described above. Alternatively, the transfer film200 is formed by doping the adhesive layer 214 with at least oneenergy-absorptive substance of the type and in the manner describedabove to a desired depth to create a gradient in the adhesive layer 214wherein the remaining un-doped portion forms the adhesive layer 214similar to the embodiment shown in FIG. 4 where the adhesive layer 214now includes retraction properties.

[0074] Referring now to FIGS. 8-9, there is shown a transfer film 400wherein the retraction properties are de-coupled from the adhesiveproperties. The transfer film 400 includes an expansion layer 412, anadhesive layer 414 and a retraction layer 430. The expansion layer 412includes a first surface 411 and a second surface 413. The expansionlayer 412 is coupled to a substrate cap 416 at the first surface 411 andto the retraction layer 430 at the second surface 413 such that theexpansion layer 412 is located between the cap 416 and the retractionlayer 430. The adhesive layer 414 includes a first surface 415 and asecond surface 417. The adhesive layer 414 is coupled to the retractionlayer 430 such that the retraction layer 430 is located between theexpansion layer 412 and the adhesive layer 414. The thickness of thetransfer film 400 is greater than approximately 10 μm with the expansionlayer 412 having a thickness of approximately 10-80 μm, the retractionlayer having a thickness of approximately 0.1-10 μm, preferably lessthan 5 μm, and the adhesive layer having a thickness of approximately0.1-10 μm preferably less than 5 μm.

[0075] The expansion layer 412 and the adhesive layer 414 is similar tothe expansion layer 112 and adhesive layer 114, respectively, asdiscussed above. The expansion layer 412 is a layer that expands whenactivated by the laser beam. The expansion layer 412 is capable ofabsorbing a sufficient amount of energy to expand and flow, and thereby,push upon a preferably un-doped retraction layer 430 and, in turn, uponthe adhesive layer 414. The retraction layer 430 is substantiallyunmelted and retains substantial elastic properties. The adhesive layer414 is preferably not activated by the laser pulse. However, theinvention is not so limited. In one embodiment, a portion of theadhesive layer 414 that is pushed by the expanding expansion layer 412and retraction layer 430 contacts the sample 418 and adheres thereto.Upon adhesion to the sample 418, the adhesive layer 414 is partiallyretracted towards the substrate cap 216 by a retractive force 226exerted by the substantially elastic retraction layer 414. Hence, theadhesive qualities and retractive qualities are separately provided bythe adhesive layer 214 and the retractive layer 430, respectively. Themechanical response of the transfer film 200 is, thereby, enhanced.After the target cells are adhered, it is desired that the transfer film400 retract from the tissue sample 418 pulling the cells out of thesample.

[0076] It is understood that the expansion layer 412 may be thermallycoupled to at least one energy-absorbing substance of the type and inthe manner discussed above. For example, thermal coupling can beaccomplished by doping the layer 412 with one or more energy absorbingsubstances or by attaching an energy-absorbing layer of the type and themanner discussed above with respect to the expansion layer 112.

[0077] The adhesive layer 414 is similar to the adhesive layer 114 ofFIGS. 2-4 and it may be thermally coupled to at least oneenergy-absorbing substance as described above and as shown in FIG. 2-4.Preferably, the adhesive layer 414 is un-doped such that it is notactivated by the laser pulse and does not substantially plasticallydeform so that it may substantially elastically retract away from thesample 418. If the laser pulse is short enough to prevent heat depositedinto the expansion layer 412 to propagate into the un-doped retractivelayer 430 and adhesive layer 414. The adhesive layer 414 and theretraction layer 430 will not melt.

[0078] From a materials standpoint, property requirements and thus thechoices are identical to other expansion layers described above. Theretraction layer 430, once again, needs to have elastomeric properties,and thus, physically and chemically cross-linked systems similar tothose described above can be utilized. Here, since the retraction layer430 does not come into actual contact with the sample, it does notrequire to have adhesive properties, except in so far as its need to beadhered to its adjacent layers. As such, tackifying agents need not tobe used in the formulation of this layer, although their use is notprecluded in the present invention. Finally, the adhesive layer 414 canbe made from any of the adhesive materials as in its counterpart,adhesive layer 114, as described in FIG. 2. In general, the softeningpoint of the adhesive layer is preferably less than the softening pointof the expansion layer.

[0079] The method of manufacturing the three-layer construction of thisembodiment of the current patent utilizes identical polymer processingtechniques to those described earlier for the two-layer counterparts. Inparticular, in one embodiment, the transfer film 400 is formed by dopingthe adhesive layer 414 with at least one energyabsorptive substance ofthe type and in the manner described above to a desired depth to createthe expansion layer wherein the remaining un-doped portion forms theretraction and adhesive layers 430, 414.

[0080] Alternatively, in another embodiment of the invention, as shownin FIG. 9, the adhesive layer 414 is shown to be coupled to theretraction layer 430 of the transfer film 400 as a doped adhesive layer414. Here, at least one concentration gradient may be formed by thedoping method; however, the invention is not so limited and theenergy-absorptive substance may be uniformly distributed throughout theexpansion layer 412 and/or the adhesive layer 414 or include a discretefilm of thermoplastic containing absorptive material thermally coupledto the adhesive layer 414 or a discrete metallic film layer thermallycoupled to the adhesive layer 414. Although FIGS. 2-9 depict thetransfer film spaced from the sample, the invention is not so limitedand contact laser micro-capture is within the scope of the presentinvention.

EXAMPLE 1

[0081] A prototype transfer film with one layer of dyed EVA (Elvax 40)as the expansion layer and one layer of nondyed polyisobutylene (Butyl065) as the adhesive layer was prepared using manual hot pressing. Eachfilm was compression molded separately, combined together, and appliedto the cap with the expansion layer facing the cap.

[0082] The EVA film was compression molded at 130° C. and 4,000 psi for3 minutes between 1 mil shims and cooled to room temperature. Thepolyisobutylene film was molded at 150° C. and 10,000 psi for 3 minutesbetween 1 mil shims and cooled to room temperature. The two films werethen combined at 70° C. and 4,000 psi for 3 minutes between 2 mil shimsand cooled to room temperature. A small diameter piece was punched outof the sheet and applied to the cap at 70° C. with light contactpressure for 3 minutes. The completed cap with laminate film was testfired 12 um above a glass slide and a wetted spot was formed thatadhered to the glass surface. Various diameters of wetted spots wereformed depending on the energy of the targeting laser.

EXAMPLE 2

[0083] A prototype transfer film with one layer of dyed EVA (Elvax 410)as the expansion layer and one layer of nondyed polyisobutylene (Butyl065) as the adhesive layer was prepared using a combination of automatedhot pressing and spin coating. The EVA film was applied to the cap usingstandard manufacturing procedures. A 10% solution of polyisobutylene wasdissolved in cyclohexane and applied to the EVA surface of the cap usinga commercial spin coater, Headway Research, Inc. The cap was held to thespinning chuck by vacuum and approximately 50 μL of polyisobutylenesolution was dispensed while the cap was spinning at 10,000 rpm. Afterspin coating, the cap was test fired 12 gm above a glass slide and awetted spot was formed that adhered to the glass surface. Variousdiameters of wetted spots were formed depending on the energy of thetargeting laser.

EXAMPLE 3

[0084] A prototype transfer film with one layer of dyed EVA (Elvax 410)as the expansion layer and one layer of block copolymer based pressuresensitive adhesive (PSA) as the adhesive layer was prepared using acombination of automated hot pressing solvent spin coating techniques.The EVA film was applied to the cap using standard manufacturingprocedures. For the adhesive layer, a polymer solution was first madefrom 10 wt/vol % of a Kraton 1107/Escorez 1310 in Toluene. Kraton 1107is a block copolymer-based (styrene-isoprene-styrene) thermoplasticrubber supplied by Kroton Polymer. Escorez 1107 is an aliphatictackifier resin supplied by Exxon Mobil Chemicals. The ratio of Kraton110 to Escorez 1310 was 1/1, based on weights. The resulting polymericsolution was then applied dynamically (as opposed to statically at thebeginning of the experiment) to the EVA surface of the cap using acommercial spin coater by Headway Research, Inc. The cap was held to thespinning chuck by vacuum and 100 μL of the polymer solution wasdispensed while the cap was spinning at 10,000 rpm and continued to spinfor 10 minutes. The acceleration and deceleration rates were both 1000rmp/sec. The resulting two-layer film has a thin (micron-level) layer ofPSA, and as such has a considerably higher tack associated with it, ascompared to the original (underlying) EVA film, which is now utilized asthe expansion layer. After spin coating, the cap was test fired 12 μmabove a glass slide and a wetted spot was formed that adhered to theglass surface. Various diameters of wetted spots were formed dependingon the energy of the targeting laser. Separately variousfrozen-sectioned tissues such as prostate, colon, and skin weresuccessfully microdissected using this 2-layer transfer film.

[0085] While the present invention has been described with reference toone or more particular variations, those skilled in the art willrecognize that many changes may be made thereto without departing fromthe spirit and scope of the present invention. Furthermore, while theinvention is described with respect to biological samples, it isunderstood that the invention is not so limited and that any sample,including non-biological samples that lend themselves to lasermicro-capture, with or without dissection, can be employed and arewithin the scope of the invention.

[0086] Each of these embodiments and obvious variations thereof arecontemplated as falling within the spirit and scope of the claimedinvention, which is set forth in the following claims.

What is claimed is:
 1. A transfer film for laser micro-capture of asample comprising: at least one expansion layer, and an adhesive layercoupled to the expansion layer; the adhesive layer being located betweenthe expansion layer and a sample for microdissection; the expansionlayer being adapted to absorb energy incident upon the transfer film andto expand to exert a force upon the adhesive layer such that a selectedportion of the sample adheres to the adhesive layer for microdissection.2. The transfer film of claim 1 wherein the expansion layer is thermallycoupled to at least one energy-absorbing substance.
 3. The transfer filmof claim 2 wherein the adhesive layer is thermally coupled to at leastone energy-absorbing substance.
 4. The transfer film of claim 3 whereinthe expansion layer and the adhesive layer are doped with at least oneindependently addressable energy absorbing substance.
 5. The transferfilm of claim 3 wherein the expansion layer and the adhesive layer aredoped with at least one spectrally selective energy-absorbing substance.6. The transfer film of claim 3 wherein the expansion layer and theadhesive layer are doped with at least one independently addressable,spectrally selective energy-absorbing substance.
 7. The transfer film ofclaim 2 wherein the at least one energy-absorbing substance is selectedfrom the group consisting of an energy-absorbing dye, a metal film, apolymer nano- composite, and Buckminsterfullerene.
 8. The transfer filmof claim 7 wherein the energy-absorbing dye is a spectrally selectivedye.
 9. The transfer film of claim 2 wherein the expansion layer isdoped with the at least one energy-absorbing substance such that atleast one concentration gradient is formed.
 10. The transfer film ofclaim 9 wherein the expansion layer includes a first surface and asecond surface; the first surface being located distally from theadhesive layer relative to the second surface; the second surface beinglocated proximately to the adhesive layer relative to the first surface.11. The transfer film of claim 10 wherein the expansion layer at thefirst surface is doped with at least one energy-absorbing substance andthe expansion layer at the second surface is not doped.
 12. The transferfilm of claim 10 wherein the expansion layer at the second surface isdoped with at least one energy-absorbing substance and the expansionlayer at the first surface is not doped.
 13. The transfer film of claim10 wherein the expansion layer is doped at the first surface and thesecond surface with the at least one energy-absorbing substance.
 14. Thetransfer film of claim 13 wherein the expansion layer is doped at thefirst surface and the second surface with at least one independentlyaddressable energy-absorbing substance.
 15. The transfer film of claim13 wherein the expansion layer is doped at the first surface and at thesecond surface with at least one spectrally selective energy-absorbingsubstance.
 16. The transfer film of claim 13 wherein the expansion layeris doped at the first surface and at the second surface with at leastone independently addressable, spectrally selective energy- absorbingsubstance.
 17. The transfer film of claim 13 wherein the expansion layeris doped at the first surface and the second surface with the sameenergy-absorbing substance.
 18. The transfer film of claim 17 whereinthe expansion layer is doped with the same concentration of theenergy-absorbing substance.
 19. The transfer film of claim 1 wherein theadhesive layer includes at least one tackifying agent.
 20. The transferfilm of claim 1 wherein the adhesive layer includes at least onepressure sensitive adhesive.
 21. The transfer film of claim 1 whereinthe adhesive layer is thermally coupled to at least one energy-absorbingsubstance.
 22. The transfer film of claim 21 wherein the at least oneenergy-absorbing substance is selected from the group consisting of anenergy-absorbing dye, a metal film, a polymer nanocomposite, andBuckminsterfullerene.
 23. The transfer film of claim 22 wherein theenergy-absorbing dye is a spectrally selective dye.
 24. The transferfilm of claim 21 wherein the adhesive layer is doped with the at leastone energy absorbing substance such that at least one concentrationgradient is formed.
 25. The transfer film of claim 24 wherein theadhesive layer includes a first surface and a second surface; the firstsurface being located proximately to the expansion layer relative to thesecond surface; the second surface being located distally to theexpansion layer relative to the first surface.
 26. The transfer film ofclaim 25 wherein the adhesive layer is doped at the first surface withthe at least one energy-absorbing substance and the adhesive layer atthe second surface is not doped.
 27. The transfer film of claim 25wherein the adhesive layer is doped at the second surface with theenergy absorbing substance and the adhesive layer at the first surfaceis not doped.
 28. The transfer film of claim 25 wherein the adhesivelayer is doped at both the first surface and the second surface with theat least one energy-absorbing substance.
 29. The transfer film of claim28 wherein the expansion layer is doped at the first surface and thesecond surface with at least one independently addressableenergy-absorbing substance.
 30. The transfer film of claim 28 whereinthe expansion layer is doped at the first surface and at the secondsurface with at least one spectrally selective energy-absorbingsubstance.
 31. The transfer film of claim 28 wherein the expansion layeris doped at the first surface and at the second surface with at leastone independently addressable, spectrally selective energy- absorbingsubstance.
 32. The transfer film of claim 28 wherein the adhesive layeris doped at both the first surface and the second surface with the sameenergy-absorbing substance.
 33. The transfer film of claim 28 whereinthe adhesive layer is doped with the same concentration of theenergy-absorbing substance.
 34. The transfer film of claim 1 wherein asoftening point of the adhesive layer is lower than a softening point ofthe expansion layer.
 35. The transfer film of claim 1 wherein asoftening point of the adhesive layer is higher than the softening pointof the expansion layer.
 36. The transfer film of claim 1 wherein theexpansion layer includes at least one thermoplastic polymer and theadhesive layer includes at least one thermoplastic polymer.
 37. Atransfer film for laser micro-capture of a sample comprising: at leastone expansion layer, and at least one adhesive layer coupled to theexpansion layer, the adhesive layer being located between the expansionlayer and a sample for microdissection; the expansion layer beingadapted to absorb energy incident upon the transfer film and to expandto exert a force upon the adhesive layer such that the adhesive layer isdeflected towards the sample and adheres to a selected portion of thesample; the adhesive layer being adapted to retract away from thesample.
 38. The transfer film of claim 37 wherein the expansion layer isthermally coupled to at least one energy-absorbing substance.
 39. Thetransfer film of claim 37 wherein the expansion layer is doped with atleast one independently addressable energy-absorbing substance.
 40. Thetransfer film of claim 37 wherein the expansion layer is doped with atleast one spectrally selective energy-absorbing substance.
 41. Thetransfer film of claim 37 wherein the expansion layer is doped with atleast one independently addressable, spectrally selectiveenergy-absorbing substance.
 42. The transfer film of claim 38 whereinthe at least one energy-absorbing substance is selected from the groupconsisting of an energy-absorbing dye, a metal film, a polymernanocomposite, and Buckminsterfullerene.
 43. The transfer film of claim42 wherein the energy-absorbing dye is a spectrally selective dye. 44.The transfer film of claim 38 wherein the expansion layer is doped withthe at least one energy-absorbing substance such that at least oneconcentration gradient is formed.
 45. The transfer film of claim 44wherein the expansion layer includes a first surface and a secondsurface; the first surface being located distally from the adhesivelayer relative to the second surface; the second surface being locatedproximately to the adhesive layer relative to the first surface.
 46. Thetransfer film of claim 45 wherein the expansion layer at the firstsurface is doped with at least one energy-absorbing substance and theexpansion layer at the second surface is not doped.
 47. The transferfilm of claim 45 wherein the expansion layer at the second surface isdoped with at least one energy-absorbing substance and the expansionlayer at the first surface is not doped.
 48. The transfer film of claim45 wherein the expansion layer is doped at the first surface and thesecond surface with the at least one energy-absorbing substance.
 49. Thetransfer film of claim 48 wherein the expansion layer is doped at thefirst surface and the second surface with at least one independentlyaddressable energy-absorbing substance.
 50. The transfer film of claim48 wherein the expansion layer is doped at the first surface and at thesecond surface with at least one spectrally selective energy-absorbingsubstance.
 51. The transfer film of claim 48 wherein the expansion layeris doped at the first surface and at the second surface with at leastone independently addressable, spectrally selective energy- absorbingsubstance.
 52. The transfer film of claim 48 wherein the expansion layeris doped at the first surface and the second surface with the sameenergy-absorbing substance.
 53. The transfer film of claim 52 whereinthe expansion layer is doped with the same concentration of theenergy-absorbing substance.
 54. The transfer film of claim 37 whereinthe adhesive layer includes at least one tackifying agent.
 55. Thetransfer film of claim 37 wherein the adhesive layer includes at leastone pressure sensitive adhesive.
 56. The transfer film of claim 37wherein a softening point of the adhesive layer is lower than asoftening point of the expansion layer.
 57. The transfer film of claim37 wherein a softening point of the adhesive layer is higher than thesoftening point of the expansion layer.
 58. The transfer film of claim37 wherein the expansion layer includes at least one thermoplasticpolymer and the adhesive layer includes at least one thermoplasticpolymer.
 59. A transfer film for laser micro-capture of a samplecomprising: at least one expansion layer, at least one retraction layercoupled to the expansion layer, and an adhesive layer coupled to theretraction layer, the adhesive layer being located between, theretraction layer and a sample for microdissection, the retraction layerbeing located between the expansion layer and the adhesive layer, theexpansion layer absorbing energy incident upon the transfer film andexpanding to exert a force upon the retraction layer and adhesive layersuch that the retraction layer and the adhesive layer are deflectedtowards the sample such that a selected portion of the sample adheres tothe adhesive layer for microdissection and the retraction layer with theattached adhesive layer retracts away from the sample.
 60. The transferfilm of claim 59 wherein the expansion layer is thermally coupled to atleast one energy-absorbing substance.
 61. The transfer film of claim 60wherein the adhesive layer is thermally coupled to at least oneenergy-absorbing substance.
 62. The transfer film of claim 61 whereinthe expansion layer and the adhesive layer are doped with at least oneindependently addressable energy absorbing substance.
 63. The transferfilm of claim 61 wherein the expansion layer and the adhesive layer aredoped with at least one spectrally selective energy-absorbing substance.64. The transfer film of claim 61 wherein the expansion layer and theadhesive layer are doped with at least one independently addressable,spectrally selective energy-absorbing substance.
 65. The transfer filmof claim 60 wherein the at least one energy-absorbing substance isselected from the group consisting of an energy-absorbing dye, a metalfilm, a polymer nanocomposite, and Buckminsterfullerene.
 66. Thetransfer film of claim 65 wherein the energy-absorbing dye is aspectrally selective dye.
 67. The transfer film of claim 60 wherein theexpansion layer is doped with the at least one energy-absorbingsubstance such that at least one concentration gradient is formed. 68.The transfer film of claim 67 wherein the expansion layer includes afirst surface and a second surface; the first surface being locateddistally from the adhesive layer relative to the second surface; thesecond surface being located proximately to the adhesive layer relativeto the first surface.
 69. The transfer film of claim 68 wherein theexpansion layer at the first surface is doped with at least oneenergy-absorbing substance and the expansion layer at the second surfaceis not doped.
 70. The transfer film of claim 68 wherein the expansionlayer at the second surface is doped with at least one energy-absorbingsubstance and the expansion layer at the first surface is not doped. 71.The transfer film of claim 68 wherein the expansion layer is doped atthe first surface and the second surface with the at least oneenergy-absorbing substance.
 72. The transfer film of claim 71 whereinthe expansion layer is doped at the first surface and the second surfacewith at least one independently addressable energy-absorbing substance.73. The transfer film of claim 71 wherein the expansion layer is dopedat the first surface and at the second surface with at least onespectrally selective energy-absorbing substance.
 74. The transfer filmof claim 71 wherein the expansion layer is doped at the first surfaceand at the second surface with at least one independently addressable,spectrally selective energy-absorbing substance.
 75. The transfer filmof claim 71 wherein the expansion layer is doped at the first surfaceand the second surface with the same energy-absorbing substance.
 76. Thetransfer film of claim 75 wherein the expansion layer is doped with thesame concentration of the energy-absorbing substance.
 77. The transferfilm of claim 59 wherein the adhesive layer includes at least onetackifying agent.
 78. The transfer film of claim 59 wherein the adhesivelayer includes at least one pressure sensitive adhesive.
 79. Thetransfer film of claim 59 wherein the adhesive layer is thermallycoupled to at least one energy-absorbing substance.
 80. The transferfilm of claim 79 wherein the at least one energy-absorbing substance isselected from the group consisting of an energy-absorbing dye, a metalfilm, a polymer nanocomposite, and Buckminsterfullerene.
 81. Thetransfer film of claim 80 wherein the energy-absorbing dye is aspectrally selective dye.
 82. The transfer film of claim 79 wherein theadhesive layer is doped with the at least one energy absorbing substancesuch that at least one concentration gradient is formed.
 83. Thetransfer film of claim 82 wherein the adhesive layer includes a firstsurface and a second surface; the first surface being locatedproximately to the expansion layer relative to the second surface; thesecond surface being located distally to the; expansion layer relativeto the first surface.
 84. The transfer film of claim 83 wherein theadhesive layer is doped at the first surface with the at least oneenergy-absorbing substance and the adhesive layer at the second surfaceis not doped.
 85. The transfer film of claim 83 wherein the adhesivelayer is doped at the second surface with the energy absorbing substanceand the adhesive layer at the first surface is not doped.
 86. Thetransfer film of claim 83 wherein the adhesive layer is doped at boththe first surface and the second surface with the at least oneenergy-absorbing substance.
 87. The transfer film of claim 86 whereinthe expansion layer is doped at the first surface and the second surfacewith at least one independently addressable energy-absorbing substance.88. The transfer film of claim 86 wherein the expansion layer is dopedat the first surface and at the second surface with at least onespectrally selective energy-absorbing substance.
 89. The transfer filmof claim 86 wherein the expansion layer is doped at the first surfaceand at the second surface with at least one independently addressable,spectrally selective energy- absorbing substance.
 90. The transfer filmof claim 86 wherein the adhesive layer is doped at both the firstsurface and the second surface with the same energy-absorbing substance.91. The transfer film of claim 86 wherein the adhesive layer is dopedwith the same concentration of the energy-absorbing substance.
 92. Thetransfer film of claim 59 wherein a softening point of the adhesivelayer is lower than a softening point of the expansion layer.
 93. Thetransfer film of claim 59 wherein a softening point of the adhesivelayer is higher than the softening point of the expansion layer.
 94. Thetransfer film of claim 59 wherein the softening point of the expansionlayer is lower than the softening point of the retraction layer.
 95. Thetransfer film of claim 59 wherein the expansion layer includes at leastone thermoplastic polymer and the adhesive layer includes at least onethermoplastic polymer; and the retraction layer includes at least onethermoplastic polymer.
 96. A transfer film for laser micro-capture of asample comprising: a first layer thermally coupled to a firstenergy-absorbing substance selected to absorb energy within a firstspectrum; a second layer coupled to the first layer such that secondlayer is proximally located to a sample for micro-capture relative tothe first layer; the second layer being thermally coupled to secondenergy-absorbing substance selected to absorb energy within a secondspectrum; wherein the first layer provides a first expansion uponactivation by at least a first laser pulse of energy within the firstspectrum to exert a force on the second layer such that a portion of thesecond layer is moved towards the sample at least a first distance; thesecond layer providing a second expansion upon activation by at least asecond laser pulse of energy within the second spectrum such that theportion of the second layer moves towards the sample a second distance.97. The transfer film of claim 96 wherein the transfer film is adaptedfor noncontact laser micro-capture.
 98. The transfer film of claim 96wherein the first and second energy-absorbing substance is selected fromthe group consisting of an energy-absorbing dye, a metal film, a polymernanocomposite, and Buckminsterfullerene.
 99. The transfer film of claim96 wherein the first spectrum does not include the second spectrum. 100.The transfer film of claim 96 wherein the first spectrum and the secondspectrum each include at least one wavelength.
 101. The transfer film ofclaim 96 wherein the second expansion temporally follows the firstexpansion.
 102. The transfer film of claim 99 wherein the second layercontacts the sample during the second expansion.
 103. The transfer filmof claim 96 wherein the first expansion temporally follows the secondexpansion.
 104. The transfer film of claim 101 wherein the second layercontacts the sample during the first expansion.